Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

Abstract

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5′-untranslated region (5′-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5′-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5′-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5′-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter. The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5′-untranslated region (5′-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5′-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5′-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5′-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter. The myeov gene was originally isolated by the application of the NIH/3T3 tumorigenicity assay with DNA from a gastric carcinoma and is activated in a subset of multiple myeloma cell lines with a t(11;14) (q13; q32) translocation (1Janssen J.W. Vaandrager J.W. Heuser T. Jauch A. Kluin P.M. Geelen E. Bergsagel P.L. Kuehl W.M. Drexler H.G. Otsuki T. Bartram C.R. Schuuring E. Blood. 2000; 95: 2691-2698PubMed Google Scholar). The chromosomal region 11q13 is frequently associated with genetic rearrangements in a large number of human malignancies, including B-cell neoplasms and carcinomas of the breast, lung, bladder, and esophagus (2Gaudray P. Szepetowski P. Escot C. Birnbaum D. Theillet C. Mutat. Res. 1992; 276: 317-328Crossref PubMed Scopus (96) Google Scholar, 3Karlseder J. Zeillinger R. Schneeberger C. Czerwenka K. Speiser P. Kubista E. Birnbaum D. Gaudray P. Theillet C. Genes Chromosomes Cancer. 1994; 9: 42-48Crossref PubMed Scopus (99) Google Scholar, 4Dickson C. Fantl V. Gillett C. Brookes S. Bartek J. Smith R. Fisher C. Barnes D. Peters G. Cancer Lett. 1995; 90: 43-50Crossref PubMed Scopus (154) Google Scholar, 5Schuuring E. Gene (Amst.). 1995; 159: 83-96Crossref PubMed Scopus (249) Google Scholar, 6Gollin S.M. Head Neck. 2001; 23: 238-253Crossref PubMed Scopus (182) Google Scholar, 7Schwab M. BioEssays. 1998; 20: 473-479Crossref PubMed Scopus (160) Google Scholar), and overexpression of myeov is frequently observed in breast tumors and oral and esophageal squamous cell carcinomas (8Janssen J.W. Cuny M. Orsetti B. Rodriguez C. Valles H. Bartram C.R. Schuuring E. Theillet C. Int. J. Cancer. 2002; 102: 608-614Crossref PubMed Scopus (40) Google Scholar). Although epigenetic effects have been demonstrated, the mechanisms of myeov regulation remain unclear (9Huang X. Gollin S.M. Raja S. Godfrey T.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11369-11374Crossref PubMed Scopus (169) Google Scholar, 10Janssen J.W. Imoto I. Inoue J. Shimada Y. Ueda M. Imamura M. Bartram C.R. Inazawa J. J. Hum. Genet. 2002; 47: 460-464Crossref PubMed Scopus (34) Google Scholar). Translational control is a final step in a complex network of regulatory processes involved in the control of gene expression. Most eukaryotic mRNAs are translated by a ribosome-scanning mechanism. The 40 S ribosomal subunit, with associated initiation factors, binds in the vicinity of the 5′ m7G-cap structure and proceeds in the 3′ direction until it encounters an initiation codon in a favorable context, and protein translation is initiated (11Kozak M. Mamm. Genome. 2002; 13: 401-410Crossref PubMed Scopus (90) Google Scholar, 12Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar, 13Kozak M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2662-2666Crossref PubMed Scopus (213) Google Scholar, 14Kozak M. Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1129) Google Scholar, 15Kozak M. Nucleic Acids Res. 2001; 29: 5226-5232Crossref PubMed Scopus (218) Google Scholar, 16Kozak M. Gene (Amst.). 2002; 299: 1-34Crossref PubMed Scopus (683) Google Scholar). Several alternative, less commonly used mechanisms of translation initiation have also been identified, including ribosome reinitiation, shunting, and internal ribosome binding. Translational initiation by internal ribosome entry involves the binding of the 40 S ribosomal subunit to an internal ribosome entry site (IRES) 3The abbreviations used are: IRESinternal ribosome entry siteCATchloramphenicol acetyltransferaseUTRuntranslated regionORFopen reading frameuORFupstream ORFssDNAsingle-stranded DNAuAUGupstream AUGFLFirefly luciferaseRLRenilla luciferaseEMCVencephalomyocarditis virus. at or near the authentic AUG. The IRES elements were first discovered in picornavirus mRNAs, where they serve to initiate translation of uncapped mRNAs (17Jackson R.J. Nature. 1988; 334: 292-293Crossref PubMed Scopus (33) Google Scholar, 18Pelletier J. Sonenberg N. Nature. 1988; 334: 320-325Crossref PubMed Scopus (1395) Google Scholar). Later, IRESs were also identified within the 5′-end of cellular mRNAs including transcripts that mediate internal initiation during the inhibition of cap-dependent translation in mitosis and apoptosis, like fgf-2, pdgf-2, igf-II, vegf, odc, c-myc, xiap, apaf-1, and bip (reviewed by Stonely and Willis (19Stoneley M. Willis A.E. Oncogene. 2004; 23: 3200-3207Crossref PubMed Scopus (291) Google Scholar) and Holcik et al. (20Holcik M. Sonenberg N. Korneluk R.G. Trends Genet. 2000; 16: 469-473Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar)). Many of the IRESs are GC-rich and contain complex secondary structures. However, no common structural features have been recognized to date. internal ribosome entry site chloramphenicol acetyltransferase untranslated region open reading frame upstream ORF single-stranded DNA upstream AUG Firefly luciferase Renilla luciferase encephalomyocarditis virus. Presently, the concept of IRES-mediated translation initiation in eukaryotes is a topic of controversial discussion (21Kozak M. Mol. Cell. Biol. 2001; 21: 1899-1907Crossref PubMed Scopus (145) Google Scholar, 22Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar, 23Kozak M. Gene (Amst.). 2003; 318: 1-23Crossref PubMed Scopus (86) Google Scholar). The gold standard for the detection of IRES activity is the dicistronic assay. In this assay, the IRES is placed between two cistrons (chloramphenicol acetyltransferase (CAT), Renilla/firefly luciferase, or other reporter genes) in a dicistronic vector and then transiently transfected into eukaryotic cells. Translation of the upstream ORF occurs via CAP-dependent scanning, whereas translation of the downstream cistron depends on the activity of the IRES. Low efficiency of translation and a lack of proper controls (e.g. Northern blotting) to exclude alternative splicing or the of a promoter within the IRES may to a of the IRES activity for a The 5′-UTR of myeov several features that mRNAs that via internal ribosome 5′-UTR has a of several upstream that may with a of and and is predicted to fold in a complex secondary structure In this we the analysis of the 5′-UTR of the myeov suggesting IRES activity were by the application of in vitro translation and RNA transfection In addition, Northern blot analysis revealed the of a cryptic promoter. we were able to show that the of the protein translation. The myeov gene the an 5′-UTR is used to translation of a protein that is to be M. Gene (Amst.). 2003; 318: 1-23Crossref PubMed Scopus (86) Google Scholar). of the and luciferase and have been and were by A. Willis M. T. Willis A.E. Nucleic Acids Res. 2000; PubMed Scopus Google Scholar). DNA was used to the myeov 5′-UTR using a cloned cDNA for the large ORF and the are were by the of and and at for The was using a and inserted into a 5′-UTR. The of the was by DNA using and The was by and and cloned into and between the and and was by the using a and a luciferase on a DNA of the promoterless constructs were by the promoter including the between the and from and by by and and and of and 5′-UTR myeov cDNA into the the of in the myeov 5′-UTR on protein DNA was used to the myeov 5′-UTR using a cloned cDNA for the large ORF and the and were with and to by with and cloned into vector, the were by DNA and of within the upstream of the myeov 5′-UTR were using as a and the as by the The were and are were by DNA and the myeov were into upstream of the Renilla luciferase cistron between the and was by DNA using a specific Renilla luciferase constructs of myeov cDNA fragments in the eukaryotic expression vector several were The open reading frame and and the in an was using codon is in and The open reading frame and and its was using and and were as were cloned into using and myeov the large and ORF were with from the vector and into In addition, the were The was by a the myeov 5′-UTR the in the was from and with and The ORF with the of myeov was from the a myeov cDNA the ORF using the with DNA and into the site of a that the the was with and with the The was to by and into the vector that was with and with the the 5′-UTR the was with and then with the The was by and and into was with and with In RNA and in and luciferase constructs were and used to in vitro transcription/translation to the were from this in and with an of and Renilla and Firefly luciferase activity were as RNA and of Translation in vitro experiments using we used a the 5′-UTR upstream of the Firefly luciferase cistron with the site and R.J. C. A. H. J.W. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In this the 5′-UTR was by the or the mutated myeov 5′-UTR using the and to in vitro constructs were with the was using the to the The and the of the mRNAs were by and of RNA were transfected into using of of a to the transfected were and with and the cell was in of were and into of and used for luciferase or first at The were and used for RNA using the direct of to the Cell and DNA were at in with and in a were into was with with and were transfected by the C. H. Mol. Cell. Biol. PubMed Scopus Google Scholar). were in and of the were with and used for luciferase assays, were used for blot analysis, and the were used for RNA were with Gene were into a The of DNA and of Gene were used for transfection to the luciferase were transfection with for at of cell were for Firefly and Renilla luciferase using the luciferase reporter and a as by the were at and in Northern cellular RNA and were isolated with the RNA and the to the Northern blot analysis and were as by et al. J.W. Bartram C.R. Oncogene. 1998; 16: PubMed Scopus Google Scholar). of RNA or of RNA were a in the of and to were in and at for with were in at by a at were to with a The were used for Firefly luciferase was by using the and Renilla was by using the for and analysis was using a to the of and with 20: PubMed Scopus Google Scholar). The the 5′-UTR of the myeov and IRES of the gene were first by using of DNA of the myeov 5′-UTR and with of in a standard during 40 we used the as a and a we used the and a of and of were then to of in a standard The single-stranded with the was in of a The at the of the was on a from the and of with a The was at and an of was RNA was isolated from that been transfected using Gene and the myeov 5′-UTR and RNA was isolated using the Gene to the analysis, of RNA were and in and in of of of single-stranded DNA was at for and at of of of and of were and for at The was with the of of 40 of and of the was in of and of was and on a DNA with a of the with the used to the single-stranded and were by and by standard were with for at were at in of and at with were with were by with a to and on a and of the myeov myeov 5′-UTR has a of and AUG upstream of the are associated with of and the of at and in the for translation M. Mamm. Genome. PubMed Scopus Google Scholar), the first show of the two whereas the AUG by a codon using the J. M. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, M. Nucleic Acids Res. 2003; PubMed Scopus Google Scholar) a strong secondary structure of this 5′-UTR with a of for the The of and secondary structure ribosome to be that myeov translation might be regulated by a (e.g. internal ribosome of the myeov 5′-UTR on Translation of to its on we cloned the myeov 5′-UTR upstream of a Firefly luciferase reporter gene into the monocistronic using the myeov AUG were transfected with and and the activity from was The were for transfection efficiency using Northern blot analysis and The of the myeov 5′-UTR upstream of the reporter gene its activity by that this gene expression In addition, we to protein in a by this an a structure was placed downstream of the and a ribosome from the 5′ site is whereas of the be transfected with this cap-dependent translation However, of the myeov 5′-UTR between the and the reporter gene luciferase activity that observed with suggest that the myeov 5′-UTR direct translation in a has been for other IRES and the of an IRES within this of IRES in the myeov 5′-UTR the the myeov 5′-UTR an we inserted it into the vector between the Renilla and reporter M. M. Willis A.E. Oncogene. 2000; PubMed Scopus Google Scholar). two reporter The first cistron is the control of an promoter and translated via a cap-dependent whereas the cistron is translated of the structure (e.g. by internal ribosome The myeov 5′-UTR was cloned upstream of the Firefly luciferase cistron were transiently transfected into and the activity of was The activity of the Firefly luciferase was to that of Renilla luciferase to in transfection that the Firefly luciferase is of the myeov 5′-UTR into the control were to alternative mechanisms as ribosomal at the Firefly luciferase initiation codon and/or the of Firefly luciferase mRNAs to alternative splicing or the of a cryptic promoter might be for this that the observed IRES activity is to ribosome reinitiation, we used the vector an upstream of the a structure in the CAP-dependent translation of the upstream cistron be whereas IRES activity of the downstream cistron be the structure expression in constructs, and In addition, activity was also in with The activity of was with with the of the myeov 5′-UTR activity by that translation initiation by the myeov 5′-UTR is on ribosome from the 5′-end of the dicistronic RNA and be to ribosome In to the the dicistronic vector also a promoter downstream of the promoter. promoter is used when constructs are in an in vitro RNA and translation using for of the RNA and a to the and 5′-UTR were in this in vitro assay In this assay, translation of also occurs via cap-dependent scanning, whereas translation of luciferase depends on the of an IRES in the Although of show that a lack of specific IRES in the for translation may to IRES activity in the myeov 5′-UTR M. T. Willis A.E. Nucleic Acids Res. 2000; PubMed Scopus Google Scholar, K. Sonenberg N. J. PubMed Google Scholar). that the observed IRES activity in the myeov 5′-UTR is to the of cryptic or in the we the promoter in the dicistronic constructs, and 5′-UTR In this no RNA and as a no translation as as activity the suggesting that a cryptic promoter is in the myeov and 5′-UTR Northern in the myeov the that experiments using monocistronic and dicistronic constructs the myeov 5′-UTR the of an IRES we also observed in vitro experiments using a transcription/translation the of and control experiments with promoterless constructs In addition, that RNA analysis be to alternative transcripts from alternative splicing and/or promoter activity when using dicistronic constructs, and this to a Northern blot activity in experiments the promoterless constructs, Northern blot analysis revealed a transcript in transfected with 5′-UTR transfected with 5′-UTR also a transcript the of a promoter in the 5′-UTR of myeov and Northern blot of other transfection experiments using 5′-UTR and 5′-UTR constructs and revealed two transcripts from the promoter and the cryptic promoter in the 5′-UTR The of the two transcripts is the and that the cryptic promoter strong Northern blot of transfected with a promoterless monocistronic the myeov 5′-UTR and of dicistronic constructs in the myeov 5′-UTR this promoter activity between the and the of the in the myeov 5′-UTR and to the initiation of the cryptic promoter in the and the IRES we were from the and to RNA of transfected with the myeov 5′-UTR and cryptic promoter activity by Northern blot analysis several initiation of the myeov cryptic promoter in the 5′-end of its 5′-UTR and The of the initiation with the cryptic promoter activity of the IRES was we were able to initiation in the 5′ of the IRES The of initiation be Translation of the strong activity of the cryptic promoter in the myeov the observed activity of 5′-UTR is with the activity of the upstream cistron suggest is strong from the cryptic promoter in the myeov translation of this transcript is In to the of the various in the myeov 5′-UTR on translation of the we several In the first we used the translation was used as an internal control for transfection The myeov 5′-UTR was inserted upstream of the In addition, we a the myeov 5′-UTR of The activity was in the vector, to when the myeov 5′-UTR was inserted upstream of the and was of show that upstream AUG triplets in the 5′-UTR of the myeov transcript control protein The of the to the of translation were using constructs of various AUG and of a be observed In to the of in the myeov 5′-UTR RNA the of effects for promoter for factors, we transiently transfected in vitro into and this we used a vector the promoter by the cistron The myeov 5′-UTR and a mutated in were were placed upstream of the were in vitro using on a with an and by a of RNA were transiently transfected into and activity was of the myeov 5′-UTR the translation efficiency of the whereas of the activity to its normal we cloned the myeov cDNA a cDNA in in the 5′-UTR were and cDNA that were the the or into the eukaryotic expression vector were transiently transfected into and RNA and protein were The were by Northern using a myeov 5′ and were by using Northern blot analysis specific transcripts of the In the first we show the analysis of a transfection in the codon of the myeov ORF was into an cloned into and transiently transfected into protein be of the normal myeov ORF with its AUG revealed two in the blot that the normal ORF of and a protein that at the AUG in the open reading in a protein with a of strong RNA expression of the two myeov cDNA constructs the and no protein be and of the in the myeov 5′-UTR protein translation of the myeov no of the 5′-UTR the translation of the RNA expression was The two experiments show that upstream AUG triplets in the 5′-UTR of the myeov transcript the expression on the translation have the 5′-UTR region of the myeov gene using several large of the that it a secondary structure, and the of several with that translation of the myeov gene might be regulated by an IRES and cellular IRESs to initiate translation in a The RNA of is uncapped and the IRES to to the vicinity of the initiation codon and translation. of cellular when cap-dependent translation is cellular translation of the mRNAs and control mechanisms of cap-dependent translation P. Genes 2001; PubMed Scopus Google Scholar). Most cellular have been identified in the mRNAs of factors, and of the myeov 5′-UTR upstream of the Firefly luciferase cistron in a monocistronic reporter vector translation a downstream of the promoter Firefly luciferase However, of the myeov 5′-UTR luciferase activity suggest that myeov translation may be initiated by an alternative (e.g. internal ribosome this we inserted the myeov 5′-UTR into the dicistronic to 5′-UTR. of the myeov 5′-UTR Firefly luciferase activity number is with the for of the other IRESs in cellular mRNAs (reviewed by (21Kozak M. Mol. Cell. Biol. 2001; 21: 1899-1907Crossref PubMed Scopus (145) Google Scholar)). that this is to the of an IRES and a of ribosomal from the first a was inserted upstream of the Renilla luciferase to the and 5′-UTR. Renilla activity that the ribosomal of the myeov 5′-UTR Firefly activity suggesting that this is to ribosomal In the dicistronic has been used to the of an IRES in a 5′-UTR. However, its is it has been recognized that the of a cryptic promoter and/or may to of the (21Kozak M. Mol. Cell. Biol. 2001; 21: 1899-1907Crossref PubMed Scopus (145) Google Scholar, 22Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar, 23Kozak M. Gene (Amst.). 2003; 318: 1-23Crossref PubMed Scopus (86) Google Scholar, P. Genes 2001; PubMed Scopus Google Scholar, B. Mol. Cell. Biol. 2002; PubMed Scopus (99) Google Scholar). several control in vitro and translation of the dicistronic the myeov of a promoterless dicistronic vector, and Northern blot of RNA from the transfected cells. in vitro transcription/translation of Firefly in the control In 5′-UTR show when with Although the of an this be by that cell IRES were in this The application of promoterless constructs the of cryptic in the cellular IRES. In this vector, the promoter with the from the dicistronic constructs is transfected into no and no translation be for the vector, 5′-UTR as as Firefly luciferase that the myeov 5′-UTR may promoter and an IRES was by Northern blot a transcript from the myeov 5′-UTR. other that that be as the of promoterless constructs, Northern and RNA when using the dicistronic (21Kozak M. Mol. Cell. Biol. 2001; 21: 1899-1907Crossref PubMed Scopus (145) Google Scholar, 22Van Eden M.E. Byrd M.P. Sherrill K.W. Lloyd R.E. RNA (N. Y.). 2004; 10: 720-730Crossref PubMed Scopus (121) Google Scholar, P. Genes 2001; PubMed Scopus Google Scholar, B. Mol. Cell. Biol. 2002; PubMed Scopus (99) Google Scholar). is also that we a transcript in RNA of transfected with a dicistronic the IRES in the is that this is to contain an IRES The the of the IRES in the midpoint of a dicistronic vector is able to function as a cryptic promoter enigmatic. we from transfected with or vector and 5′-UTR and 5′-UTR dicistronic constructs the myeov 5′-UTR in the region an The that this transcript to a Renilla luciferase and also been when using promoterless constructs that it from the myeov 5′-UTR. the of a promoter as identified by Northern of transfected with and dicistronic constructs, of several 5′ of cDNA cDNA fragments as as analysis of human the of myeov transcripts suggest that in its normal context, this promoter is used at or used at a specific developmental in a specific cell or apoptosis, using dicistronic constructs, and transfection of mutated and fragments of the myeov cDNA that translation of myeov is by the of and of the revealed that a on translation. of efficiency by the of is for cell and translation efficiency is regulated (11Kozak M. Mamm. Genome. 2002; 13: 401-410Crossref PubMed Scopus (90) Google Scholar, Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). is the where of in the from the of within a specific of C. A. E. J. 2003; PubMed Scopus Google Scholar). region two in the mechanisms may also of the The of has been regulation of and may on the developmental stage or as for the gene C. A. E. J. 2003; PubMed Scopus Google Scholar). with other myeov RNA mRNAs may in 5′-UTR and in a (8Janssen J.W. Cuny M. Orsetti B. Rodriguez C. Valles H. Bartram C.R. Schuuring E. Theillet C. Int. J. Cancer. 2002; 102: 608-614Crossref PubMed Scopus (40) Google Scholar). of in the 5′-UTR of and a promoterless revealed strong promoter activity B. Mol. Cell. Biol. 2002; PubMed Scopus (99) Google Scholar). is in with and that it is of to the that is IRES activity in cellular In addition, we that translation of protein is regulated by in the 5′-UTR. regulation of and its function Willis for the and The of is